The overall goal of this proposal is to provide the first molecular insights into H3K4 demethylation by the JARID1 family of oncoproteins. The JARID1/KDM5 family of proteins are H3K4me2/3 specific lysine demethylases. Biochemically the JARID1 proteins are members of the jumonji C (JmjC) family of proteins that demethylate methylated lysines through the use of -ketoglutarate and Fe(II) as cofactors. However, unlike many of the JmjC proteins, the JARID1 proteins contain multiple auxiliary functional domains (JmjN, ARID, zinc finger and three PHD) proximal to the JmjC domain that contribute to protein functions including protein stability, catalysis and substrate specificity. Another unique aspect about the JARID1 family of proteins is their association in disease. For example, JARID1B is upregulated in a variety of cancers including breast, lung, prostate, bladder and melanoma. Downregulation of JARID1B in tumor cells also leads to growth suppression, indicating that this enzyme is an ideal target in multiple cancers. To understand how regions outside the catalytic JmjC domain of JARID1 contribute to protein stability, catalysis and substrate binding specificity we propose to prepare recombinant JARID1 deletion constructs. These constructs will be used for purification and enzyme kinetics studies to establish the contribution of each of the JmjC proximal domains by employing the use of previously developed histone demethylase activity assays. In addition, we propose to determine the three-dimensional crystal structure of the regions of JARID1B that are necessary and sufficient for H3K4 demethylation. This structure will identify the relative importance of key amino acid residues whose role in catalysis and substrate binding will be determined using site-directed mutagenesis, a demethylation assay and isothermal calorimetry experiments in order to carry out a structure-function analysis of JARID1B activity. In addition, known inhibitor compounds of JARID1 will be tested using a histone demethylase activity assay against purified protein. These studies will be used to determine the mode of inhibition (competitive, noncompetitive, uncompetitive) of the inhibitors. JARID1 will be cocrystallized with these inhibitors for crystal structure determination. These experiments will provide a molecular framework for the structure-based design of potent and selective JARID1 inhibitors. The combined studies described above will provide the first molecular insights into H3K4me2/3 demethylation by the JARID1 family of proteins. The structure/function relationships that will be discovered will also provide critical information forthe design of JARID1-selective inhibitors that will aid in the development of drugs for anti-cancer therapy.